Annotation of novel neuropeptide precursors in the migratory locust based on transcript screening of a public EST database and mass spectrometry

BACKGROUND: For holometabolous insects there has been an explosion of proteomic and peptidomic information thanks to large genome sequencing projects. Heterometabolous insects, although comprising many important species, have been far less studied. The migratory locust Locusta migratoria, a heterometabolous insect, is one of the most infamous agricultural pests. They undergo a well-known and profound phase transition from the relatively harmless solitary form to a ferocious gregarious form. The underlying regulatory mechanisms of this phase transition are not fully understood, but it is undoubtedly that neuropeptides are involved. However, neuropeptide research in locusts is hampered by the absence of genomic information. RESULTS: Recently, EST (Expressed Sequence Tag) databases from Locusta migratoria were constructed. Using bioinformatical tools, we searched these EST databases specifically for neuropeptide precursors. Based on known locust neuropeptide sequences, we confirmed the sequence of several previously identified neuropeptide precursors (i.e. pacifastin-related peptides), which consolidated our method. In addition, we found two novel neuroparsin precursors and annotated the hitherto unknown tachykinin precursor. Besides one of the known tachykinin peptides, this EST contained an additional tachykinin-like sequence. Using neuropeptide precursors from Drosophila melanogaster as a query, we succeeded in annotating the Locusta neuropeptide F, allatostatin-C and ecdysis-triggering hormone precursor, which until now had not been identified in locusts or in any other heterometabolous insect. For the tachykinin precursor, the ecdysis-triggering hormone precursor and the allatostatin-C precursor, translation of the predicted neuropeptides in neural tissues was confirmed with mass spectrometric techniques. CONCLUSION: In this study we describe the annotation of 6 novel neuropeptide precursors and the neuropeptides they encode from the migratory locust, Locusta migratoria. By combining the manual annotation of neuropeptides with experimental evidence provided by mass spectrometry, we demonstrate that the genes are not only transcribed but also translated into precursor proteins. In addition, we show which neuropeptides are cleaved from these precursor proteins and how they are post-translationally modified

Peptidomic survey of the locust neuroendocrine system

Neuropeptides are important controlling agents in animal physiology. In order to understand their role and the ways in which neuropeptides behave and interact with one another, information on their time and sites of expression is required. We here used a combination of MALDI-TOF and ESI-Q-TOF mass spectrometry to make an inventory of the peptidome of different parts (ganglia and nerves) of the central nervous system from the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. This way, we analysed the brain, suboesophageal ganglion, retrocerebral complex, stomatogastric nervous system, thoracic ganglia, abdominal ganglia and abdominal neurohemal organs. The result is an overview of the distribution of sixteen neuropeptide families, i.e. pyrokinins, pyrokinin-like peptides, periviscerokinins, tachykinins, allatotropin, accessory gland myotropin, FLRFamide, (short) neuropeptide F, allatostatins, insulin-related peptide co-peptide, ion-transport peptide co-peptide, corazonin, sulfakinin, orcokinin, hypertrehalosaemic hormone and adipokinetic hormones (joining peptides) throughout the locust neuroendocrine system.status: publishe

Coupling matters - A combination of liquid chromatography and mass spectrometry for peptidomic research

The guest authors describe the use of LC-MS for the analysis of peptides. They deal with peptide extraction, peptidomic analysis in organisms with and without genomic databases, identification of peptides with conserved N- and C- terminal sequences, and in situ MALDI-TOF peptide profiling.status: publishe

Conservation of fruitless’ role as master regulator of male courtship behaviour from cockroaches to flies

In Drosophila melanogaster, male courtship behaviour is regulated by the fruitless gene. In D. melanogaster, fruitless encodes a set of putative transcription factors that are sex-specifically spliced. Male-specific variants are necessary and sufficient to elicit male courtship behaviour. Fruitless sequences have been reported in other insect species, but there are no data available on their functional role. In the present work, we cloned and sequenced fruitless in males of the German cockroach, Blattella germanica, and we studied its expression in male brain and testes. B. germanica fruitless encodes a 350-amino acid protein with BTB and Zinc finger domains typical of fruitless sequences. Upon RNAi-mediated knockdown of fruitless in B. germanica, males no longer exhibit courtship behaviour, thus implying that fruitless is necessary for male sexual behaviour in our cockroach model. This suggests that the role of fruitless as master regulator of male sexual behaviour has been conserved along insect evolution, at least from cockroaches to flies.Financial support from the Ministry of Education and Science, Spain (projects BFU2008-00484 to M-D. Piulachs and CGL2008-03517/BOS to X. Bellés), Generalitat de Catalunya (2005 SGR 00053) is gratefully acknowledged. L. Ciudad received a pre-doctoral research grant (I3P) from CSIC, and E. Clynen received a travel grant from the Fund for Scientific Research (FWO)-Flanders (Belgium) to work in the Institute of Evolutionary Biology in Barcelona. Thanks are also due to Elena Torres, who received a JAE-intro (CSIC) grant, for helping in the experimental work.Peer reviewe

A review of the most important classes of serine protease inhibitors in insects and leeches

The constant increase of life expectancy is associated with major aging of developed populations. This indicates that the new century will have one of most epidemic progressions of cardiovascular, cancer and inflammatory diseases. The high challenge for medical research is to compress such morbidity. Invertebrates have demonstrated to be truly useful models in drug discovery for such aging diseases. The last decade, drug discovery in leeches has opened the gate for new molecules to treat emphysema, coagulation, inflammation, dermatitis and cancer. Also other invertebrates, such as insects, harvest potential interesting molecules, such as serine protease inhibitors that can be exploited by the medical industry. In this review we discuss the most important classes of serine protease inhibitors in insects and leeches. © 2005 Bentham Science Publishers Ltd.status: publishe

Trypsin and chymotrypsin inhibitors in insects and gut leeches

The constant increase of life expectancy is associated with major ageing of developed populations. This indicates that the new century will have one of most epidemic progressions of cardiovascular, cancer and inflammatory diseases. The high challenge for medical research is to compress such morbidity. In these conditions, invertebrates have demonstrated to be truly useful models in drug discovery for such ageing diseases. The last decade, drug discovery in leeches has opened the gate of new molecules to treat emphysema, coagulation, inflammation, dermitis and cancer. Also other invertebrates such as insects, which evolved from the annelids, harvest potential interesting molecules, such as serine protease inhibitors that can be exploited by the medical industry.status: publishe

New insights into the evolution of the GRF superfamily based on sequence similarity between the locust APRPs and human GRF

In a manual sequence alignment experiment we accidentally observed that the locust adipokinetic hormone (AKH)(1) precursor-related peptides (APRPs), which are peptides contained in the AKH-preprohormones, display sequence similarity with human growth hormone-releasing factor (GRF). So far, a counterpart for GRF, either structurally or functionally, has not yet been reported in any non-chordate. This could give new insights into the origin of the GRF superfamily, as it would mean that in some invertebrates (locusts), the structural homologues of glucagons (AKHs) and GRFs (APRPs) are located on the same precursor. No function could as yet be attributed to APRPs, although these neuropeptides occur abundantly in the corpora cardiaca, a neurohaemal organ from which many neuropeptides are released. Therefore, similar to GRF, a role as releasing factor may be envisaged for APRPs. (C) 2004 Elsevier Inc. All rights reserved.status: publishe

Identification of new members of the (short) neuropeptide F family in locusts and Caenorhabditis elegans

Both the long and short neuropeptides F (NPF) represent important families of invertebrate neuropeptides that have been implicated in the regulation of reproduction and feeding behavior. In the present study, two short NPFs (SNRSPS(L/I)R(L/I)RFamide and SPS(L/I)R(L/I)RFamide) were de novo sequenced by mass spectrometry in two major pest insects, the desert locust Schistocerca gregaria and the African migratory locust Locusta migratoria. They are two of the most widespread peptides in the locust neuroendocrine system. A peptide that was previously reported to accelerate egg development in S. gregaria is shown to represent a truncated form of long NPF. This peptide is most likely derived by a novel processing mechanism involving cleavage at RY. In addition, an NPF peptide from the nematode Caenorhabditis elegans was isolated and sequenced by tandem mass spectrometry.status: publishe

The use of peptidomics in endocrine research. Minireview

In 2002, the Nobel Prize for chemistry was awarded to the inventors of two novel ionization techniques in mass spectrometry, MALDI and ESI. These techniques, often in combination with data from genomic databases, represent an extremely powerful tool in analytical (bio)chemistry, with many applications, e.g., in the field of proteomics. Peptides, which are small proteins, have, despite their importance as controlling agents in numerous physiological processes, as yet been much less intensively studied by these novel techniques than larger proteins. The term peptidomics, i.e., the study of all peptides expressed by a certain cell, organ or organism was only introduced in 2001. In neuroendocrinology, spectacular progress could already be realized and the future looks bright. In this minireview we discuss the different methodologies that are used in peptidomics and give an overview of the wide range of applications. (C) 2003 Elsevier Science (USA). All rights reserved.status: publishe

Peptide differential display: a novel approach for phase transition in locusts

Today, the question of the physiological cause of phase transition, the transition from the solitary to the gregarious phase, in locusts remains unanswered. We hereby present a novel approach by which we have attempted to determine whether different phases express or release different peptides in similar physiological conditions. For this purpose, a peptidomic analysis of the corpora cardiaca and hemolymph of crowded and isolated locusts of Schistocerca gregaria was performed using high performance liquid chromatography and matrix-assisted laser desorption ionisation time of flight mass spectrometry. A comparison between the two conditions reveals differences in the number and amount of peptides present in the corpora cardiaca and the hemolymph. Further research will have to identify these phase specific differences and their role in locust phase polymorphism. (C) 2002 Elsevier Science Inc. All rights reserved.status: publishe